The optic tectum, the primary visual center in non-mammalian vertebrates, receives retinal fibers in a topographically ordered manner. en (en-1 and en-2, homologs of the Drosophila segment polarity gene engrailed) is expressed in the tectal primordium in a rostrocaudal gradient, around the stage when the polarity of the retinotectal projection map is being determined. Here we report that scattered en expression, caused by retroviral gene transfer, perturbed the retinotopic order. Nasal retinal fibers, which normally recognize the caudal side of the tectum (strong en expression side) as a target, arborized at ectopic sites, as if they found their targets, or degenerated. Temporal retinal fibers, which normally recognize the rostral side of the tectum (weak en expression side) as a target, were also affected in some cases by degeneration or prevention of innervation in the tectum. These results suggest that gradient en expression defines the positional identity of the tectum along the rostrocaudal axis.